1. Field of the Invention
The present invention relates to host cells useful in the production of recombinant proteins. In particular, the invention relates to non-toxic, non-toxigenic, and non-pathogenic fungal host cells of Fusarium which can be used in the high-level expression of recombinant proteins, especially enzymes. The invention further relates to promoter and terminator sequences which may be used in such a system.
2. Description of the Related Art
The use of recombinant host cells in the expression of heterologous proteins has in recent years greatly simplified the production of large quantities of commercially valuable proteins, which otherwise are obtainable only by purification from their native sources. Currently, there is a varied selection of expression systems from which to choose for the production of any given protein, including prokaryotic and eukaryotic hosts. The selection of an appropriate expression system will often depend not only on the ability of the host cell to produce adequate yields of the protein in an active state, but also to a large extent may be governed by the intended end use of the protein.
Although mammalian and yeast cells have been the most commonly used eukaryotic hosts, filamentous fungi have now begun to be recognized as very useful as host cells for recombinant protein production. Examples of filamentous fungi which are currently used or proposed for use in such processes are Neurospora crassa, Acremonium chrysogenum, Tolypocladium geodes, Mucor circinelloides and Trichoderma reesei, Aspergillus nidulans, Aspergillus niger and Aspergillus oryzae.
Certain species of the genus Fusarium have been used as model systems for the studies of plant pathogenicity and gene regulation such as Fusarium oxysporum (Diolez et al., 1993, Gene 131:61-67; Langin et al., 1990, Curr. Genet. 17:313-319; Malardier et al., 1989, Gene 78:147-156 and Kistler and Benny, 1988, Curr. Genet. 13:145-149), Fusarium solani (Crowhurst et al., 1992, Curr. Genet. 21:463-469), and Fusarium culorum (Curragh et al., 1992, Mycol. Res. 97:313-317). These Fusarium sp. would not be suitable commercially for the production of heterologous proteins because of their undesirable characteristics such as being plant pathogens or because they produce unsafe levels of mycotoxin. Dickman and Leslie (1992, Mol. Gen. Genet. 235:458-462) discloses the transformation of Gibberella zeae with a plasmid containing nit-2 of Neurospora crassa. The strain of Gibberella zeae disclosed in Dickman and Leslie is a plant pathogen and produces zearalenone, an estrogenic mycotoxin. Sanchez-Fernandez et al. (1991, Mol. Gen. Genet. 225:231-233) discloses the transformation of Gibberella fujikoroi carrying a niaD mutation with a plasmid containing the Aspergillus niger niaD gene.
An ideal expression system is one which is substantially free of protease and mycotoxin production, also substantially free of large amounts of other endogenously made secreted proteins, and which is capable of higher levels of expression than known host cells. The present invention now provides new Fusarium expression systems which fulfill these requirements.